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Mechanisms in the cellular defense against oxidative stress

Corbin, M.V.

2017

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Corbin, M. V. (2017). Mechanisms in the cellular defense against oxidative stress.

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Chapter 3

Loss of NARFL function deregulates iron regulatory and

cohesin complex cleavage proteins under

hyperoxia-induced oxidative stress

M.V. Corbin1_{; D.A.P Rockx}1_{; T.V. Pham}2_{; S.R. Piersma}2_{; J.C. Knol}2_{; H. Joenje}1_;

C.R. Jimenez2_{*; J.C.Dorsman}1_*

1_{Department of Clinical Genetics, Cancer Center Amsterdam,}

VU University Medical Center, 1081 HV Amsterdam, The Netherlands

2_{Department of Medical Oncology, Cancer Center Amsterdam,}

Abstract

Introduction

Oxidative damage of macromolecules such as DNA, lipid and proteins has been associated with numerous age-related diseases including cancer and neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease (Alhebshi et al., 2012). Extensive research has been conducted to establish disease-causative mechanisms; nevertheless, a full understanding of these mechanisms is still not complete. Identifying genes and cellular networks that are essential for the defense and/or prevention against/of oxidative stress and its subsequent damage could provide novel targets that could aid in the treatment of certain age-related diseases.

We have recently identified nuclear prelamin A recognition factor-like (NARFL) as a crucial factor involved in the defense against hyperoxia-induced oxidative stress in human cells. The study comparing hyperoxia-resistant cell lines with their sensitive counterparts revealed amplification of the NARFL-containing chromosomal region accompanied by overexpression at both mRNA and protein levels in the resistant cells. Knockdown of NARFL in the hyperoxia-resistant cell lines increased their susceptibility to hyperoxia, which was manifested by a decrease of cell viability under hyperoxic stress. We also observed that under hyperoxic stress the NARFL-depleted cell lines exhibited an impairment in the cohesion of sister-chromatids, suggesting a role for NARFL in the maintenance of chromosomal structure under oxidative stress (Figure 1) (Corbin et al., 2015).

To date, NARFL has been primarily associated with the highly conserved cytosolic

iron-sulfur protein assembly (CIA) pathway. It has been reported that NARFL plays a role in the maturation of iron-sulfur (Fe-S) clusters, which are subsequently inserted by complexes such as MMS19/CIA complex into apoproteins that function within the cytosol and the nucleus (Seki et al., 2013; Song and Lee, 2011, 2008). The Fe-S cluster-containing proteins are involved in many biological processes such as DNA metabolism, translation, RNA modification and regulation of gene expression (Mettert and Kiley, 2015; Kimura and Suzuki,

2015; Gari et al., 2012; Stehling et al., 2012; Klinge et al., 2007; Rudolf et al., 2006). Fe-S

clusters are co-factors that have been shown to be extremely sensitive to reactive oxygen species (ROS), becoming highly unstable when exposed to these reactive oxygen molecules

(Py et al., 2011; Imlay, 2006). The loss of these co-factors has been shown to render some

Fe-S proteins inactive and thereby unable to carry out the various functions needed by the biological processes with which they have been associated.

for cell survival. Other studies in Saccharomyces cerevisiae and Caenorhabditis elegans have also linked the NARFL counterparts, Nar1/Narp1 to oxygen sensitivity and a possible association with lifespan has been reported (Zhao et al., 2014; Fujii et al., 2009). Although the evidence for the involvement of NARFL in the CIA pathway as well as the defense against ROS or oxidative stress is undisputed, the underlying associated and/or affected biological processes are still poorly understood.

Figure 1: Illustration of the hyperoxia adaptation process and NARFL protein levels of the sensitive cells to

Material and Methods Cell lines

The hyperoxia-sensitive cell lines HeLa-20_NT and Hela-20_shNARFL, as well as the hyperoxia-resistant cell lines HeLa-80_NT and HeLa-80_shNARFL were routinely cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and Sodium Pyruvate (Life Technologies Europe BV, The Netherlands) at

normoxia (20% O₂, 75% N₂, 5% CO₂).

Cell cycle analysis

The non-targeted cell lines HeLa-20_NT and HeLa-80_NT as well as their stable NARFL knockdown counterparts Hela-20_shNARFL and HeLa-80_shNARFL were used for cell

cycle analysis. Cells were seeded in 75 cm2 _{flasks and cultured overnight at 20% oxygen. A}

selection of flasks was shifted to hyperoxic stress (80% oxygen) for 48 h and 72 h and the remaining flasks were kept at 20% oxygen, as controls. Cells were harvested, washed in

Phosphate Buffer Saline (PBS) solution and fixed in ice-cold 70% EtOH. Cells were washed

and resuspended in PBS with 1:10 PI/RNase staining buffer (BD Biosciences) and analyzed by flow cytometry on a BD FACSCalibur (BD Biosciences). Cell-cycle analysis was conducted with BD CellQuest software (BD Biosciences).

Sample preparation proteome analysis

Gel electrophoresis, in-gel digestion and mass spectrometry

Equal amount of proteins (Supplementary Figure 1) were separated on NuPAGE™ Novex™ 12% Bis-Tris gels (Life Technologies Europe BV, The Netherlands). Gels were stained with Coomassie brilliant blue G-250 and subsequently destained with milli-Q water. Gel lanes were sliced into five bands followed by in-gel digestion with trypsin (Promega) (Shevchenko et al., 1996). Peptide extraction and MS analysis have been previously described (Piersma et al., 2010). In brief, peptides were extracted once in 1% formic acid and afterwards twice in 50% acetonitrile/5% formic acid. The peptide extract volume was reduced in a vacuum centrifuge and subsequently separated by an Ultimate 3000 nanoLC system (Dionex LC-Packings, The Netherlands) equipped with a 20 cm × 75 µm ID fused silica column custom packed with 3 µm 120 Å ReproSil Pur C18 aqua (Dr Maisch GMBH, Germany). Intact peptide MS spectra and MS/MS spectra were acquired and masses were measured.

Data analysis

Protein selection. To identify top NARFL-associated protein candidates under hyperoxia,

protein-protein interaction (PPI) network of the selected differential proteins using the

Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and visualized with Cytoscape software (3.2.0) (Szklarczyk et al., 2015; Shannon et al., 2003; Snel et al., 2000).

Enrichment analysis. To determine which gene ontology (GO) categories were significantly

Figure 2: Flow cytometry cell cycle analysis of HeLa-20_NT, HeLa-20_shNARFL, HeLa-80_NT, and HeLa-80_shNARFL under

hy-peroxic stress and normoxic condition. NT= non-targeted

Results

NARFL depletion and hyperoxia affects cell cycle progression

As NARFL knockdown was shown to affect sister-chromatid cohesion, an essential process in cell division, we first wanted to investigate the effects of NARFL depletion combined with hyperoxia on cell cycle progression. A complete disruption of the cell cycle profile of the HeLa-20_NT after exposure to hyperoxia was observed as compared to the same cell line under normoxic condition. We observed an increased sub-G1 peak, indicating the appearance of apoptotic cells, with increasing hyperoxia exposure. There was also a decrease of the G1-phase and an increase in the S and G2/M peak after hyperoxia exposure. This is in contrast to the HeLa-80_NT cell line, where no apparent hyperoxic stress effect could be observed (Figure 2A and Supplementary Figure 2). However, upon NARFL knockdown an accumulation in the G2/M-phase was observed upon hyperoxia exposure of HeLa-80_shNARFL cells, suggesting a delay or arrest in that particular cell cycle phase.

0 10 20 30 40 50 60 70 80 G1 S G2/M

HeLa-20_NT HeLa-20_shNARFL HeLa-80_NT HeLa-80_shNARFL

Regulation of CIA pathway components as result of hyperoxia adaptation

To further investigate the underlying cellular mechanism(s) that could explain the correlation between NARFL and processes such as sister-chromatid cohesion/genomic maintenance, cell cycle regulation and defense against oxidative stress, we performed proteome analysis. We only selected the hyperoxia-resistant cell lines, since the acquired overexpression of NARFL in these cells was of significance for their survival under hyperoxia/ oxidative stress (Figure 3).

Figure 3: Illustration, effect of NARFL protein level modification under normoxic and hyperoxic conditons on biological processes

and cell survival. Top: under normoxia, no detectable effect on NARFL-linked biological processes in NARFL knockdown cells compared to non-targeted cells. Bottom: under hyperoxia, impaired NARFL-linked biological processes (sister-chromatid cohesion, cell cycle) in NARFL knockdown cells compared to the non-targeted counterpart.

hyperoxia-resistant

Hyperoxia

non-targeted NARFLNARFL

1: NARFL link ed pr ot eins under h yper oxia 2: Hyper oxia r esponse pr ot eins, KD NARFL 3: Hyper oxia r esponse pr ot eins, NT NARFL 4: NARFL link ed pr ot

eins under normo

xia CIA pa th w ay R FC p KD h yp KD h yp NT hy pNT hy pR FC p KD h yp KD h yp KD nor KD nor R FC p NT hy pNT hy pNT nor NT nor R FC p KD nor KD nor NT nor NT nor CIAPIN1 -1.2 0.57 16 16 13 14 -1.0 0.92 16 16 18 14 --1.2 0.46 13 14 16 17 --1.1 0.79 18 14 16 17 NUBP1 --1.7 0.37 3 1 3 4 -1.0 0.98 3 1 2 2 --1.1 0.80 3 4 4 4 --2.1 0.24 2 2 4 4 NDOR1 --100 0.24 0 0 1 0 --100 0.24 0 0 0 1 -100 0.24 1 0 0 0 -100 0.25 0 1 0 0 NARFL --100 0.09 0 0 1 1 -1.0 1.00 0 0 0 0 ↓ -7.5 0.01 1 1 6 9 ↓ -100 0.001 0 0 6 9 CIA O1 --1.0 0.98 6 5 5 6 --1.0 0.90 6 5 4 7 --1.3 0.57 5 6 6 8 --1.2 0.63 4 7 6 8 FAM96B -1.4 0.45 6 8 4 6 --1.1 0.91 6 8 8 6 -1.1 0.83 4 6 5 4 -1.6 0.28 8 6 5 4 FAM96A --1.4 0.57 2 3 4 3 --1.2 0.80 2 3 3 3 -2.4 0.22 4 3 1 2 -1.9 0.36 3 3 1 2 MMS19 -1.0 0.89 5 9 8 6 --1.1 0.91 5 9 9 6 --1.1 0.86 8 6 9 6 -1.0 0.94 9 6 9 6 Fe-S prot eins ABCE1 ↓ -1.6 0.01 28 26 46 40 --1.3 0.19 28 26 38 31 -1.1 0.72 46 40 44 38 --1.2 0.28 38 31 44 38 DP YD -1.0 1.00 0 0 0 0 --100 0.14 0 0 0 2 --100 0.24 0 0 1 0 -1.8 0.62 0 2 1 0 ER CC2 --100 0.24 0 0 0 1 --100 0.24 0 0 1 0 --3.1 0.32 0 1 2 1 --3.1 0.30 1 0 2 1 RTEL1 -1.0 1.00 0 0 0 0 --100 0.24 0 0 1 0 -1.0 1.00 0 0 0 0 -100 0.25 1 0 0 0 POLD1 --1.5 0.20 8 11 12 16 ↓ -2.2 0.01 8 11 18 25 --1.6 0.07 12 16 19 25 --1.0 0.86 18 25 19 25 ACO 1 ↓ -2.3 0.05 4 4 10 8 ↓ -4.8 0.01 4 4 25 13 --1.8 0.08 10 8 21 12 -1.2 0.62 25 13 21 12 CHTF18 -1.3 0.33 13 18 11 13 -1.1 0.83 13 18 13 17 --1.2 0.60 11 13 10 17 -1.1 0.81 13 17 10 17 NTHL1 ↓ -100 0.04 0 0 1 2 ↓ -100 0.002 0 0 8 5 --2.4 0.22 1 2 3 4 -1.8 0.23 8 5 3 4 PRIM2 --1.2 0.60 7 8 11 7 --1.7 0.12 7 8 13 12 --1.4 0.25 11 7 16 10 --1.0 0.90 13 12 16 10 FC= F old Chang e; R= r egula tion; P: p-value; NT h yp: HeLa-80_NT (h yper oxia); KD h yp: HeLa-80_shNARFL (h yper

oxia); KD nor: HeLa-80_shNARFL (normo

xia); NT nor: HeLa-80_NT (normo

xia). Comparison 1: HeLa-80_shNARFL (h yper oxia) v s. HeLa-80_NT (h yper

oxia); Comparison 2: HeLa-80_shNARFL (h

yper

oxia) v

s. HeLa-80_shNARFL (normo

xia); Comparison 3: HeLa-80_NT (h

yper

oxia)

vs. HeLa-80_NT (normo

xia); Comparison 4: HeLa-80_shNARFL (normo

xia) v s. HeLa-80_NT (normo xia). Table I: Iden tified CIA pa th w ay pr ot eins and F e-S pr ot

To obtain an overview of the data an unsupervised hierarchical clustering analysis using all identified proteins was performed. Within the two batches the clustering corresponds to the presence or absence of NARFL (Figure 4).

Since NARFL is known as an essential component of the CIA pathway, we first performed a targeted analysis of the other components of this pathway, as well as known cytosolic and nuclear Fe-S proteins. NARFL was the only CIA component that was observed as down-regulated, due to its experimental knockdown and as a result of its response to hyperoxia exposure. In addition to NARFL, 4 Fe-S proteins were identified as deregulated. The proteins ACO1 (↓) and NTHL1 (↓) were down-regulated in the hyperoxia-resistant HeLa-80 cells under hyperoxia in a NARFL-dependent manner (comparison 1), while these were also down-regulated in NARFL depleted resistant cells upon shifting to hyperoxia (comparison 2). In contrast, the proteins ABCE1 (↓) and POLD1 (↓) were only found to be down-regulated in one of the two conditions, respectively (Table I).

Figure 5: A) Sample comparison design for proteomics data analysis (left panel). NARFL counts (normalized) from

proteomics data set for respective comparisons. B) Differential proteins of main comparison HeLa-80_NT (80% O2) vs.

HeLa-80_shNARFL (80% O2) [top panel]. Differential proteins reference comparisons HeLa-80_shNARFL (20% O2) vs.

HeLa-80_shNARFL (80% O2), HeLa-80_NT (20% O2) vs. HeLa-80_NT (80% O2), and HeLa-80_NT (20% O2) vs. HeLa-80_

shNARFL (20% O2) [middle panel]. Prioritization criteria for the NARFL linked candidates [bottom panel].

B

Main

comparison HeLa - 80_NT (80% O2) vs. HeLa-80_shNARFL (80% O2):_{222 differen�al proteins: 115↓ and107↑ (total protein in data 5465)} 1: NARFL associated proteins under hyperoxia

Re

fe

rence

comparisons

HeLa - 80_NT (20% O2) vs. HeLa-80_shNARFL (20% O2): 191 differen�al proteins: 104 ↓ and 87↑

HeLa - 80_shNARFL (20% O2) vs. HeLa-80_shNARFL (80% O2): 110 differen�al proteins: 73 ↓ and 37↑

2: Hyperoxia response proteins with depleted NARFL

(total protein in data 5432)

4: NARFL associated proteins under normoxia

(total protein in data 5510)

Top ranked: Mid ranked: Protein rank ing criteria A

Cell lines comparison design HeLa-80_NT

(80% oxygen) HeLa-80_shNARFL (80% oxygen)

HeLa-80_shNARFL (20% oxygen) 1 2 3 HeLa-80_NT (20% oxygen) 4 Comparison 1 Comparison 4 Comparison 2 Comparison 3

HeLa - 80_NT (20% O2) vs. HeLa-80_NT (80% O2): 56 differen�al proteins: 37 ↓ and 19 ↑

3: Hyperoxia response proteins with NARFL (NT)

(total protein in data 5433)

NT_ ( hyp) KD_(hy p) KD_(nor) KD_(hy p) NT_ ( hyp) NT_ ( nor) NT_ ( nor) KD_(nor) NARFL counts C1 C2 C3 C4 ↓ ↑ ↓ ↓ C1 C2 C3 C4 ↑ ↑ ↓ ↑ ↓

_ _ _

_ _ _

Table II: Do wn-r egula ted pr ot eins in HeLa-80_shNARFL c ompar ed t

o HeLa-80_NT and their r

egula tion in r ef er ence c omparisons 1: NARFL link ed pr ot eins under h yper oxia 2: Hyper oxia r esponse pr ot eins, KD NARFL 3: Hyper oxia r esponse pr ot eins, NT NARFL 4: NARFL link ed pr ot

eins under normo

Mid Con tinue t able II UQCR C1 ↓ -1.5 0.02 30 32 50 42 --1.3 0.13 30 32 47 34 -1.1 0.52 50 42 43 41 --1.0 0.80 47 34 43 41 DP YSL3 ↓ -1.5 0.02 40 33 55 53 -1.0 0.94 40 33 37 35 -1.1 0.60 55 53 52 47 --1.4 0.04 37 35 52 47 XR CC5 ↓ -1.5 0.002 94 100 143 142 --1.1 0.30 94 100 103 113 -1.1 0.38 143 142 144 121 --1.2 0.05 103 113 144 121 FC= Fold Chang e; R= regula tion; P: p-value; NT hyp: HeLa-80_NT (h yper oxia); KD hyp: HeLa-80_shNARFL (h yper oxia); KD nor: HeLa-80_shNARFL (normo xia); NT nor: HeLa-80_NT (normo xia). Comparison 1: HeLa-80_shNARFL (h yper oxia) v s. HeLa-80_NT (h yper oxia); Comparison 2: HeLa-80_shNARFL (h yper oxia) vs. HeLa-80_shNARFL (normo xia); Comparison 3: HeLa-80_NT (h yper oxia) vs. HeLa-80_NT (normo

xia); Comparison 4: HeLa-80_shNARFL (normo

xia) v

s. HeLa-80_NT (normo

1: NARFL link ed pr ot eins under h yper oxia 2: Hyper oxia r esponse pr ot eins, KD NARFL 3: Hyper oxia r esponse pr ot eins, NT NARFL 4: NARFL link ed pr ot

eins under normo

xia Symbol R FC p KD h yp NT hy p R FC p KD h yp KD nor R FC p NT hy p NT nor R FC p KD nor NT nor Top CA V1 ↑ 100 0.05 2 1 0 0 ↑ 100 0.04 2 1 0 0 --100 0.10 0 0 1 1 --100 0.09 0 0 1 1 HPS5 ↑ 100 0.05 1 2 0 0 ↑ 100 0.04 1 2 0 0 -1.0 1.00 0 0 0 0 -1.0 1.00 0 0 0 0 PT TG 1 ↑ 100 0.01 3 2 0 0 ↑ 100 0.01 3 2 0 0 --100 0.24 0 0 1 0 --100 0.23 0 0 1 0 NE CTIN2 ↑ 100 0.05 1 2 0 0 ↑ 100 0.04 1 2 0 0 --100 0.15 0 0 2 0 --100 0.15 0 0 2 0 UBE2J1 ↑ 100 0.05 2 1 0 0 ↑ 100 0.04 2 1 0 0 --100 0.10 0 0 1 1 --100 0.09 0 0 1 1 COL12A1 ↑ 5.6 0.01 7 9 1 2 ↑ 5.8 0.01 7 9 1 2 -2.9 0.32 1 2 1 0 -2.8 0.34 1 2 1 0 MDN1 ↑ 2.0 0.01 29 41 15 19 ↑ 1.7 0.04 29 41 17 25 -1.1 0.64 15 19 15 16 -1.4 0.21 17 25 15 16 ABHD16A ↑ 100 0.05 2 1 0 0 -1.0 0.98 2 1 2 1 ↓ -100 0.01 0 0 2 4 --2.0 0.31 2 1 2 4 IR GQ ↑ 100 0.02 2 2 0 0 -1.4 0.69 2 2 1 2 ↓ -100 0.02 0 0 2 2 --1.4 0.68 1 2 2 2 MRPS21 ↑ 100 0.02 2 2 0 0 -1.0 0.96 2 2 3 1 ↓ -100 0.04 0 0 1 2 -1.3 0.76 3 1 1 2 TMEM57 ↑ 100 0.05 1 2 0 0 -1.0 0.94 1 2 0 3 ↓ -100 0.03 0 0 1 3 --1.4 0.66 0 3 1 3 WDR89 ↑ 100 0.02 2 2 0 0 --1.4 0.58 2 2 2 4 ↓ -100 0.03 0 0 3 1 -1.4 0.58 2 4 3 1 TMEM14C ↑ 3.7 0.02 6 9 2 2 -1.5 0.31 6 9 5 5 ↓ -3.0 0.05 2 2 7 5 --1.3 0.62 5 5 7 5 CENPF ↑ 1.9 0.01 37 37 18 21 --1.1 0.61 37 37 34 46 ↓ -1.7 0.03 18 21 27 39 -1.2 0.27 34 46 27 39 CA CUL1 ↑ 100 0.02 2 2 0 0 -1.0 0.97 2 2 2 2 -1.0 1.00 0 0 0 0 ↑ 100 0.02 2 2 0 0 NMI ↑ 100 0.01 3 2 0 0 --1.3 0.62 3 2 2 5 -1.0 1.00 0 0 0 0 ↑ 100 0.01 2 5 0 0 CPLX1 ↑ 8.0 0.04 2 6 0 1 -2.0 0.25 2 6 3 1 -100 0.24 0 1 0 0 ↑ 100 0.03 3 1 0 0 COL7A1 ↑ 7.6 0.02 3 5 1 0 -1.0 0.96 3 5 3 5 -100 0.24 1 0 0 0 ↑ 100 0.005 3 5 0 0 GDA ↑ 4.6 0.01 9 13 1 4 -1.1 0.82 9 13 10 11 --1.4 0.57 1 4 3 4 ↑ 3.0 0.01 10 11 3 4 PHLDB2 ↑ 3.9 0.03 4 8 1 2 --1.1 0.89 4 8 5 7 -1.5 0.67 1 2 1 1 ↑ 6.2 0.01 5 7 1 1 DS T ↑ 3.8 0.003 19 15 5 4 -1.0 0.99 19 15 13 21 -1.5 0.46 5 4 2 4 ↑ 5.8 0.004 13 21 2 4

Table III: Up-r

egula ted pr ot eins in HeLa-80_shNARFL c ompar ed t

o HeLa-80_NT and their r

Con tinue t able III Mid SUP T5H ↑ 1.6 0.02 35 39 21 25 -1.2 0.43 35 39 29 35 --1.1 0.77 21 25 21 28 -1.3 0.16 29 35 21 28 CKAP5 ↑ 1.6 0.005 71 78 44 49 -1.2 0.18 71 78 56 69 -1.1 0.73 44 49 40 48 -1.4 0.03 56 69 40 48 HBD ↑ 1.6 0.04 26 29 14 20 -1.1 0.62 26 29 25 25 --1.4 0.12 14 20 26 24 --1.0 1.00 25 25 26 24 STUB1 ↑ 1.5 0.03 47 40 24 33 -1.0 0.81 47 40 42 42 --1.0 0.86 24 33 24 35 -1.4 0.05 42 42 24 35 SUP T6H ↑ 1.5 0.04 29 36 21 22 -1.2 0.32 29 36 28 26 -1.0 1.00 21 22 18 24 -1.3 0.29 28 26 18 24 FC= Fold Ch ang e; R= regula tion; P: p-value; NT hyp: HeLa-80_NT (h yper oxia); KD hyp: HeLa-80_shNARFL (h yper oxia); KD nor: HeLa-80_shNARFL (normo xia); NT nor: HeLa-80_NT (normo xia). Comparison 1: HeLa-80_shNARFL (h yper oxia) v s. HeLa-80_NT (h yper oxia); Comparison 2: HeLa-80_shNARFL (h yper oxia) vs. HeLa-80_shNARFL (normo xia); Comparison 3: HeLa-80_N T (h yper oxia) vs. HeLa-80_NT (normo

xia); Comparison 4: HeLa-80_shNARFL (normo

xia) v

s. HeLa-80_NT (normo

Prioritization of NARFL linked proteins under oxidative stress

It is under hyperoxic stress, that we observed the most significant difference between cells that still have a functioning level of NARFL (non-targeted cells) and cells that have lost this ability (NARFL knockdown cells) (Corbin et al., 2015). Therefore, NARFL functions will be most optimally identified in NARFL knockdown in HeLa-80 cells under hyperoxia compared to their non-targeting counterparts (comparison 1: HeLa-80_shNARFL versus

HeLa-80_NT both at 80% O₂). The differential proteins of the other comparisons (2-4)

uncover NARFL (in) dependent regulation and aids in prioritization the differential proteins of comparison 1 (Figure 5A). Top candidates have the same regulation in comparison 2 and/ or 4 and the opposite profile or less severe response in comparison 3 compared to NARFL knockdown HeLa-80 cells under hyperoxia (comparison 1) (Figure 5B). In total 222 proteins were identified as differentially expressed in response to NARFL depletion under hyperoxic stress (comparison 1), with 115 down-regulated and 107 up-regulated proteins in NARFL knockdown HeLa-80 cells under hyperoxia (Figure 5B, Table II and III respectively). Proteins

with the same regulation in comparison 2 [HeLa-80_shNARFL (80% O₂) versus HeLa-80_

shNARFL (20% O₂)] were considered to be the most interesting candidates, since these

proteins are deregulated as a result of NARFL depletion, combined with hyperoxic response similar to comparison 1. A total of 110 proteins were identified as differentially expressed in comparison 2 of which 24 were also differential in the same direction (17 ↓ and 7 ↑) as comparison 1; thus consisting of prime candidates (Figure 5B, 6A, Table II and III). When compared to the hyperoxia response reference comparison (comparison 3) [HeLa-80_NT

(80% O₂) versus HeLa-80_NT (20% O₂)], proteins of interests are the 8 overlapping proteins

in the opposite direction (1 ↓ and 7 ↑) to comparison 1 (Figure 5B, 6B, and Table II and III). NARFL linked proteins of HeLa-80 cells kept under normoxia regulated in the same direction as in cells kept under normoxia regulated in the same direction as in cells kept under hyperoxia are also of interest. With this third reference comparison, (comparison 4),

[HeLa-80_shNARFL versus HeLa-80_NT both at 20% O₂], we found 51 overlapping proteins that

NARFL depletion combined with hyperoxic stress affects cell division and iron metabolism associated proteins

1: NARFL linked proteins

under hyperoxia 2: Hyperoxia response proteins with depleted NARFL

98 proteins 100 proteins 17 proteins 7 proteins 0 proteins 0 proteins 1 2 1 2 1 2 1 2 1 2 1 2 84 198 24

1: NARFL linked proteins under hyperoxia

3: Hyperoxia response proteins in presence of NARFL 2 proteins 0 proteins 112 proteins 100 proteins 1 proteins 7 proteins 1 3 1 3 1 3 1 3 1 3 1 3 46 212 10 A B

1: NARFL linked proteins

under hyperoxia 4: NARFL linked proteins under normoxia

84 proteins 31 proteins 0 proteins

1 4 1 4 1 4 140 171 51 C

87 proteins 20 proteins 0 proteins

Figure 6: Prioritization of NARFL associated differentially expressed proteins of the main comparison 1 [HeLa-80_NT (80% O2) vs.

HeLa-80_shNARFL (80% O2)] in correlation with comparison 2 [HeLa-80_shNARFL (20% O2) vs. HeLa-80_shNARFL (80% O2)] (A),

comparison 3 [HeLa-80_NT (20% O2) vs. HeLa-80_NT (80% O2)] (B) and comparison 4 [HeLa-80_NT (20% O2) vs. HeLa-80_shNARFL

Figur e 7: Pr ot ein-pr ot ein in ter action (PPI) ne tw ork of select ed do wn-r egula ted pr ot eins in c omparison 1: HeLa-80_NT (h yper oxia) vs. HeLa-80_shNARFL (h yper oxia) and their associa ted enriched gene on tology (GO) ca teg ories. Node siz e: Fold chang e; Node color: pr ot ein ranking; Node shape: ov erlap be tw een main comparison and re fer ence comparisons. Comparison 2: HeLa-80_shNARFL (normo xia) vs. HeLa-80_shNARFL (h yper oxia). Comparison 3: HeLa-80_NT (normo xia) vs. HeLa-80_NT (h yper oxia). Comparison 4: HeLa-80_NT (normo xia) vs. HeLa-80_shNARFL (normo xia). Solid line: con fidence sc or e 0.7- 0.9. Dashed line: c on fidence sc or

e 0.4-0.6. (See also figur

e 9, f or associa ted GO) SNCA ARRB1 MCAM CYP1B1 XRCC5 LIG4 NAT1 DARS EIF5 GTF2B KLF5 FARSB RRN3 NDUFV1 ERGIC1 NTHL1 PGM2L1 NDUFV3 NDUFAF5 NDUFS1 PFKL UQCRC1 ACO1 NOA1 OAS3 PMPCB MRPS7 PNPT1 NSUN4 PLA2G4A MAVS MVP MCM5 MCM6 STAT1 TIMP1 DNAJA2 HSPA13 MAP2 AGO1 DYNC1I2 NQO2 COG2 GCLC EARS2 GLUL PPAT ABCE1 GSPT1 HARS2 PARVA CTNNAL1 DPH1 CRAT ASPH GPR56 AKR1C2 WASH3P RASA3 MANF RIN1 MUC13 CHID1 CLK3 SRSF4 AKR1C1 PMM2 ANKMY2 NAPRT ZNF143 RETSAT SNCG SRP72 OTUD7B MAP2K7 DPYSL3 GALK2 FAM160A2 RALGAPA2 KCMF1 SAPCD2 TMEM38B LRCH1 IREB2 THAP11 ARMCX3 VPS18 _TMA16 ZKSCAN8 PLEKHA1 KANSL2 AKR1C3 NUCB1 BAIAP2L1 RBMS1 PTGR1 DERL1 MRPL9 FAM83H QSOX2 WASH6P MAGEA1 ARFGAP3 STK39 EPPK1 LRRC41 NSUN5 AUP1 B4GALT3 MTDH SQRDL TRPS1 ARHGEF40 CHKA PDLIM3 Node color

Overlap comparison 2 Overlap comparison 3 Overlap comparison 4

Node size - 1.5 - 100 Node shape FC 0.05 0.001 p

confidence score: 0.7-0.9 confidence score: 0.4-0.6

25

Figure 8: GO enrichment analysis. GO terms associated with overrepresented down-regulated proteins. According to fold

enrichment (FE) score high → low. BP= Biological Processes; MF= Molecular Function; CC= Cellular Components.

0 20 40 60 80 100 120

response to iron(II) ion doxorubicin metabolic process cellular response to prostaglandin D s�mulus nega�ve regula�on of interleukin -8 produc�on re�nal metabolic process regula�on of cellular respira�on establishment of integrated proviral latency endosome organiza�on tRNA aminoacyla�on for protein transla�on response to heat G1/S transi�on of mito�c cell cycle protein homooligomeriza�on aging oxida�on -reduc�on process trans -1,2-dihydrobenzene -1,2 -diol dehydrogenase ac�vity 4 iron, 4 sulfur cluster binding alpha-tubulin binding NADH dehydrogenase (ubiquinone) ac�vity double -stranded DNA binding oxidoreductase ac�vity magnesium ion binding poly(A) RNA binding nonhomologous end joining complex MCM complex rough endoplasmic re�culum nuclear chromosome, telomeric region mitochondrial inner membrane early endosome cytoskeleton mitochondrion cytosol membrane FE Count

Enriched categories down-regulated proteins

Figur e 9: Pr ot ein-pr ot ein in ter action (PPI) ne tw ork of select ed up-r egula ted pr ot eins in comparison 1: HeLa-80_NT (h yper oxia) vs. HeLa-80_shNARFL (h yper oxia) and their associa ted enriched gene on tology (GO) ca teg ories. Node siz e: Fold chang e; Node color: p-value; Node shape: ov erlap be tw een main comparison and re fer ence comparisons. Comparison 2: HeLa-80_shNARFL (normo xia) vs. HeLa-80_shNARFL (h yper oxia). Comparison 3: HeLa-80_NT (normo xia) vs. HeLa-80_NT (h yper oxia). Comparison 4: HeLa-80_NT (normo xia) vs. HeLa-80_shNARFL (normo xia). Solid line: con fidence sc or e 0.7- 0.9. Dashed line: con fid ence sc or e

0.4-0.6. (See also figur

e 10, f or associa ted GO) SIN3B ATM MED1 EIF4EBP 1 ANAPC16 AMOT DST MTCL1 CKAP5 INCENP CENPF CLIP1 TSC2 BIRC5 IGF2BP3 PTTG1 ESPL1 CAV1 TACC1 NFKB2 BCL2L1 NMI MED9 CDKN2C EDRF1 MUC1 TNS3 _CDH2 MET HLTF SUPT6H NECTIN2 STUB1 RNF20 UBE2J1 TRAPPC2L SUPT5H ANKRD54 ZNF629 LSM2 MTR DECR2 PRRC2A CCDC144A IRGQ SLC9A3R2 ABHD16A ZDHHC5 AQR IFIT1 MDN1 LIMA1 ZNF280C FAM129A ATP5I ZNF66 TMEM57 C1QTNF3 TMPO ARID1A DPH6 C1orf174 NPEPPS HELZ UACA WDR89 GDA TRIO MRPS21 IDH2 C3 HBD SH3D19 FTH1 COL7A1 COL12A1 MRPS24 MRPS21 POMK CHMP7 TRIP11 EIF1 CPLX1 PIGP RPP38 GBF1 DIAPH3

PHLDB2 HAUS3 _ISCU SPECC1L

TMEM14C RAD51AP1 ZFC3H1 NFX1 CACUL1 VPS50 ENSA ALDH16A1 HPS5 RWDD1 TRAPPC2L WNK1 GTF2E2 CEP97 EMSY ZNF646 ZNF598 UBE3C

Overlap comparison 2 Overlap comparison 3 Overlap comparison 4

Node size 1.5 100 Node shape FC Node color 0.05 0.003 p

confidence score: 0.7-0.9 confidence score: 0.4-0.6

2

1

Figure 10: GO enrichment analysis. GO terms associated with overrepresented up-regulated proteins. According to fold enrichment

(FE) score high → low. BP= Biological Processes; MF= Molecular Function; CC= Cellular Components.

0 20 40 60

homologous chromosome segrega�on nega�ve regula�on of mRNA polyadenyla�on cytokinesis cellular lipid metabolic process regula�on of mito�c cell cycle microtubule cytoskeleton organiza�on nega�ve regula�on of protein binding sister chroma�d cohesion mito�c nuclear division protein ubiqui�na�on involved in ubiqui�n-dependent protein catabolic process regula�on of cell cycle G2/M transi�on of mito�c cell cycle cell cycle arrest protein polyubiqui�na�on cell division cell -cell adhesion DNA repair transcrip�on from RNA polymerase II promoter potassium channel inhibitor ac�vity

protein complex scaffold phosphatase binding ubiqui�n protein ligase ac�vity cadherin binding involved in cell -cell adhesion poly(A) RNA binding protein homodimeriza�on ac�vity protein binding microtubule plus -end intermediate filament cytoskeleton basal plasma membrane cell leading edge kinetochore chromosome, centromeric region spindle midbody microtubule cytoskeleton nuclear envelope cell -cell adherens junc�on protein complex centrosome cytosol membrane nucleus Count FE

Enriched categories up-regulated proteins

Discussion

The objective of this study was to further investigate NARFL function(s) associated with its role in survival against hyperoxia-induced oxidative stress through identification of NARFL linked proteins. In the hyperoxia-resistant cell line HeLa-80, NARFL depletion and hyperoxic stress resulted in cell cycle disruption compared to its non-targeted counterpart, with an accumulation of cells in the G2/M-phase. The down-regulated proteins as a result of NARFL knockdown and hyperoxic stress were predominantly associated with biological processes such as response to iron (II) ion, doxorubicin metabolic process, and regulation of cellular respiration. The identified up-regulated proteins were annotated to biological processes such as cell cycle and homologous chromosome segregation.

With the unsupervised clustering analysis we, nevertheless, observed a possible batch effect with the technical replicates, as they clustered separately. This could well be a processing effect. In theory this could, of course, lead to identification of differential proteins that might not be relevant to the research question(s) being investigated. Nonetheless, preliminary results of the other HeLa cell line (HeLaS3) have also shown differential expression for some of the proteins, such AKR1C3, AKR1C2, AKR1C1, further validating the proteins discussed.

be of significance for this observation might be the proteins annotated to the molecular function double-strand DNA binding namely, the down-regulated proteins XRCC5, STAT1, RBMS1, and NTHL1. The latter is a NARFL linked top ranked Fe-S protein that is undetectable in NARFL-knockdown cells in hyperoxia in comparisons 1 and 2, which might have been affected by the loss of NARFL function related to the CIA pathway. Nonetheless, the possible relevance of NTHL1 down-regulation in the accumulation of cell in the G2-phase is not clear as a previous study has suggested that activity of this protein does not influence DNA repair of cells delayed in the G2 phase when exposed to ionizing radiation (Mjelle et al., 2015; Chaudhry, 2007). Thus, the known cell division associated proteins that were identified does not provide a clear answer for the observed accumulation of cells in the G2-phase.

The biological process response to iron (II) ion (ACO1, SNCA, IREB2) had the highest fold enrichment score in the GO enrichment analysis of the down-regulated proteins. The protein ACO1 has been shown to have dual function which depends on the presence of

Fe-S clusters (Philpott et al., 1994). Bound by a Fe-S cluster allows ACO1 to carry out its

aconitase function catalyzing the conversion of citrate to isocitrate. When the Fe-S cluster is lost due to oxidative stress or decrease iron levels, ACO1 is known as IRP1 and binds to iron responsive element (IRE) containing proteins and thereby regulates iron homeostasis

(Meyron-Holtz et al., 2004). IREB2/IRP2 is also essential for iron homeostasis and together

The down-regulated proteins of the aldo-keto reductase family, AKR1C3, AKR1C2, AKR1C1 were associated with many of the identified enriched biological processes. These

proteins belong to a group of enzymes involved in the conversion of aldehydes and ketones

into alcohols. These proteins have been shown to catalyze the conversion of prostaglandins

into different end products (Lanisnik Rizner, 2012; Ebert et al., 2011). AKR1C3 has been

previously linked to ROS removal, as elevated levels have been shown to lead to increased resistance to ionizing radiation (Xiong et al., 2014). Additionally, elevated AKR1C3 and AKR1C1 levels have been shown to lead to increased resistance to cisplatin in colon cancer cells (Ebert et al., 2011). Increased drug resistance has also been suggested for increased

AKR1C2 levels (Ebert et al., 2011). It could be suggested that NARFL aids in maintaining

functional AKR1C proteins levels which in turn plays a role in the removal of ROS, at least in the case of AKR1C3. The AKR1C proteins have also been associated with Nuclear factor erythroid 2-related factor 2 (NF2L2), a well documented transcription factor that regulates antioxidant genes that contain antioxidant response element (ARE). It has been shown that AKR1C proteins also contain ARE. A study has shown that the AKR1C proteins are inducible

by NF2L2 which ultimately led to detoxification of the lipid peroxide 4HNE (Jung and Kwak,

2013). Further investigation into a possible role of NARFL in NF2L2 activity is warranted to

determine if the observed NARFL effect is independent of this activity.

We have previously shown that NARFL depletion and hyperoxic stress led to increased sister chromatid cohesion defects. GO enrichment analysis have identified several proteins that might explain this observation. The proteins CKAP5, INCENP, CLIP1, CENPF, and BIRC5 were specifically annotated to the biological process sister chromatid cohesion. These proteins were up-regulated due to NARFL depletion and hyperoxic stress; however the disadvantageous effect of this is not completely clear. Expected is that in order to explain the increased cohesion defects, proper maintenance of chromosomal segregation would be compromised when NARFL is depleted and under hyperoxic stress. This argument does not seem to hold true for either of these proteins as their expression is essential for chromosomal alignment and segregation. The up-regulated proteins ESPL1 and PTTG1 were annotated to the biological process homologous chromosome segregation. It has been well documented that ESPL1 is essential for sister chromatid separation during the metaphase-to-anaphase transition, as it cleaves the cohesin subunit SCC1, releasing the centromeric cohesion complexes. The activity of ESPL1 is regulated by Cdk1/cyclin-B1 as well as PTTG1/securin, however the role of the latter in maintenance of chromosome integrity is contentious.

Overexpression or deregulated ESPL1 leads to improper sister chromatid segregation (Haaß

et al., 2015; Hellmuth et al., 2015; Pfleghaar et al., 2005). It could be suggested that up

cohesion defects previously observed.

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Figure 1: Schematic representation of experimental setup (top) Cells are seeded on day 0 and cultured overnight at normoxia

(20% oxygen). On day 1, cells are subset of the cells was shifted to hyperoxia (80/95% oxygen) for 48 h, while the rest remain at normoxia. On day 3 the cells were harvested. (Bottom) Coomassie stained gel.

Supplementary data

Day 0 Day 1

48 hours

48 hours

Day 3

1: HeLa-80_NT (20% oxygen [A]) 2: HeLa-80_NT (80% oxygen [A]) 3: HeLa-80_shNARFL (20% oxygen [A]) 4: HeLa-80_shNARFL (80% oxygen [A]) 5: HeLa-80_NT (20% oxygen [B]) 6: HeLa-80_NT (80% oxygen [B]) 7: HeLa-80 _shNARFL (20% oxygen [B]) 8: HeLa-80_shNARFL (80% oxygen [B])

Term p-value FE C Genes

Biologic

al Pr

ocess

response to iron(II) ion 6.16E-4 76.3 3 ACO1, SNCA, IREB2

doxorubicin metabolic process 1.14E-3 57.2 3 AKR1C3, AKR1C2, AKR1C1

cellular response to prostaglandin D

stimulus 3.83E-2 50.9 2 AKR1C3, AKR1C2

negative regulation of interleukin-8

production 4.46E-2 43.6 2 ARRB1, OTUD7B

retinal metabolic process 2.64E-3 38.2 3 AKR1C3, CYP1B1, AKR1C1

regulation of cellular respiration 5.08E-2 38.2 2 PNPT1, NOA1

establishment of integrated proviral

latency 5.08E-2 38.2 2 XRCC5, LIG4

endosome organization 1.83E-2 14.3 3 FAM160A2, VPS18, WASH3P

tRNA aminoacylation for protein

translation 2.78E-2 11.4 3 DARS, HARS2, FARSB

response to heat 6.34E-3 10.5 4 PLA2G4A, GCLC, MAP2K7, DNAJA2

G1/S transition of mitotic cell cycle 2.88E-2 6.0 4 GSPT1, MCM5, PPAT, MCM6

protein homooligomerization 2.93E-2 4.3 5 GLUL, DERL1, PNPT1, DPYSL3, AKR1C1

aging 4.40E-2 3.7 5 PLA2G4A, GCLC, SNCA, IREB2, TIMP1

oxidation-reduction process 5.03E-3 2.8 11 AKR1C3, AKR1C2, PTGR1, UQCRC1, CYP1B1, SNCA, ASPH, QSOX2, AKR1C1, NQO2, RETSAT

Molecular Function

trans-1,2-dihydrobenzene-1,2-diol

dehy-drogenase activity 2.63E-4 111.2 3 AKR1C3, AKR1C2, AKR1C1

4 iron, 4 sulfur cluster binding 8.48E-6 21.2 6 ACO1, NDUFV1, IREB2, NTHL1, NDUFS1, PPAT

alpha-tubulin binding 1.49E-2 15.9 3 WASH6P, SNCA, WASH3P

NADH dehydrogenase (ubiquinone)

activity 4.09E-2 9.3 3 NDUFV3, NDUFV1, NDUFS1

double-stranded DNA binding 1.93E-2 7.0 4 XRCC5, STAT1, NTHL1, RBMS1

oxidoreductase activity 2.36E-3 5.1 7 AKR1C3, SQRDL, AKR1C2, PTGR1, SNCA, AKR1C1, RETSAT

magnesium ion binding 5.08E-2 3.6 5 GLUL, GCLC, SNCA, FARSB, MAP2K7

poly(A) RNA binding 4.71E-4 2.5 19 XRCC5, MTDH, EPPK1, DARS, PNPT1, EIF5, MRPL9, MRPS7, NOA1, MANF, NDUFV3, SRSF4, GSPT1, CLK3, HARS2, AGO1, SRP72, NSUN5, RBMS1

Table I: Enriched biological functions annotated to the down-regulated proteins Continue table I

Cellular Componen

t

nonhomologous end joining complex 4.82E-2 40.3 2 XRCC5, LIG4

MCM complex 5.40E-2 35.8 2 MCM5, MCM6

rough endoplasmic reticulum 4.81E-2 8.5 3 NUCB1, GLUL, SNCA

nuclear chromosome, telomeric region 4.63E-2 5.0 4 XRCC5, LIG4, MCM5, MCM6

mitochondrial inner membrane 1.61E-3 3.7 10 NDUFV3, SQRDL, NDUFAF5, PLA2G4A, UQCRC1, NDUFV1, MRPL9, NOA1, CRAT, MRPS7

early endosome 5.44E-2 3.5 5 NUCB1, WASH6P, VPS18, DERL1, WASH3P

cytoskeleton 8.87E-3 3.4 8 CTNNAL1, EPPK1, BAIAP2L1, RIN1, STK39, FAM83H, PARVA, MVP

mitochondrion 1.25E-3 2.3 19 MAVS, ABCE1, NDUFAF5, CYP1B1, UQCRC1, ACO1, PNPT1, SNCA, IREB2, MRPL9, CRAT, NOA1, MRPS7, EARS2, NDUFV3, GLUL, HARS2, NTHL1, NDUFS1

cytosol 1.46E-5 1.9 41 CTNNAL1, XRCC5, CHKA, ARFGAP3, GCLC, SNCA, EIF5,

OAS3, DPH1, PPAT, AKR1C3, WASH6P, AGO1, STK39, RASA3, MAP2K7, MUC13, AKR1C1, DNAJA2, FAM160A2, PFKL, DARS, ACO1, BAIAP2L1, NAT1, IREB2, DPYSL3, STAT1, PMM2, NAPRT, PGM2L1, GLUL, PLA2G4A, GSPT1, ARRB1, FARSB, WASH3P, SRP72, DYNC1I2, COG2, PARVA

membrane 1.43E-2 1.7 23 XRCC5, ABCE1, ARFGAP3, DERL1, PFKL, DARS, LRRC41, PNPT1, SNCA, ERGIC1, MCM5, TMEM38B, NUCB1, CHID1, CLK3, FARSB, ASPH, NDUFS1, AUP1, RETSAT, DNAJA2, MVP, COG2

Term p-value FE C Genes

Biologic

al pr

ocess

homologous chromosome

segre-gation 3.49E-2 56.0 2 ESPL1, PTTG1

negative regulation of mRNA

polya-denylation 4.62E-2 42.0 2 SUPT5H, RNF20

cytokinesis 2.84E-3 14.0 4 INCENP, BIRC5, ESPL1, BCL2L1 cellular lipid metabolic process 2.21E-2 12.9 3 SIN3B, DECR2, MED1 regulation of mitotic cell cycle 2.21E-2 12.9 3 GBF1, BIRC5, RNF20 microtubule cytoskeleton

organi-zation 8.17E-4 11.8 5 SPECC1L, BIRC5, DST, PHLDB2, TACC1 negative regulation of protein binding 4.46E-2 8.8 3 CAV1, IFIT1, STUB1

sister chromatid cohesion 3.11E-3 8.2 5 CKAP5, INCENP, CLIP1, CENPF, BIRC5

mitotic nuclear division 1.15E-4 6.0 9 ANAPC16, HAUS3, CKAP5, INCENP, CLIP1, CENPF, BIRC5, ENSA, PTTG1

protein ubiquitination involved in ubiquitin-dependent protein catabolic process

1.28E-2 5.5 5 ANAPC16, CACUL1, UBE3C, PTTG1, STUB1 regulation of cell cycle 3.70E-2 5.4 4 TSC2, CENPF, ATM, MED1

G2/M transition of mitotic cell cycle 4.82E-2 4.9 4 HAUS3, CKAP5, BIRC5, ENSA cell cycle arrest 5.35E-2 4.7 4 CDKN2C, TSC2, DST, ATM

protein polyubiquitination 2.31E-2 4.6 5 UBE3C, NPEPPS, HLTF, RNF20, STUB1

cell division 1.01E-3 4.4 9 SPECC1L, ANAPC16, HAUS3, CKAP5, CENPF, BIRC5, ENSA, PTTG1, TACC1

cell-cell adhesion 2.24E-2 3.7 6 LIMA1, CKAP5, DIAPH3, TMPO, SLC9A3R2, PHLDB2 DNA repair 5.04E-2 3.6 5 EMSY, RAD51AP1, PTTG1, STUB1, ATM

transcription from RNA polymerase

II promoter 3.32E-2 2.6 8 GTF2E2, NMI, PTTG1, NFKB2, HLTF, SUPT5H, TRIP11, NFX1

Molecular Function

potassium channel inhibitor activity 4.05E-2 48.1 2 WNK1, ENSA protein complex scaffold 2.31E-2 12.6 3 ISCU, CAV1, SLC9A3R2 phosphatase binding 2.76E-2 11.5 3 TSC2, WNK1, SLC9A3R2

ubiquitin protein ligase activity 2.50E-2 4.5 5 CACUL1, UBE2J1, HLTF, STUB1, MED1 cadherin binding involved in cell-cell

adhesion 2.83E-2 3.5 6 LIMA1, CKAP5, DIAPH3, TMPO, SLC9A3R2, PHLDB2 poly(A) RNA binding 2.39E-3 2.4 16 MRPS24, PRRC2A, HELZ, MRPS21, IGF2BP3, HLTF, ZFC3H1,

GTF2E2, AQR, ZNF598, MTCL1, EIF1, LSM2, SUPT5H, SUPT6H, NFX1

protein homodimerization activity 2.81E-2 2.3 10 EMSY, TSC2, CLIP1, CENPF, NECTIN2, BIRC5, MTCL1, BCL2L1, DST, STUB1

protein binding 3.76E-4 1.5 54 HPS5, NFKB2, PTTG1, GTF2E2, SIN3B, ANKRD54, EIF4EBP1, GBF1, CDKN2C, INCENP, LSM2, SUPT5H, FAM129A, NECTIN2, ESPL1, ARID1A, HLTF, SLC9A3R2, TACC1, ISCU, TNS3, MED9, CLIP1, RNF20, DST, SUPT6H, MED1, CEP97, C1ORF174, CAV1, NMI, CACUL1, C3, BCL2L1, UBE3C, IGF2BP3, STUB1, FTH1, COL7A1, TRIP11, HBD, MUC1, RAD51AP1, CKAP5, MET, TRIO, CENPF, BIRC5, ATM, IFIT1, UACA, TSC2, RWDD1, AMOT

Table II: Enriched biological functions annotated to the up-regulated proteins Continue table II

Cellular Componen

t

microtubule plus-end 3.93E-3 31.5 3 CKAP5, CLIP1, DST

intermediate filament cytoskeleton 1.97E-4 17.2 5 DST, MDN1, PHLDB2, STUB1, TACC1 basal plasma membrane 1.52E-2 15.7 3 CAV1, MET, DST

cell leading edge 2.07E-2 13.4 3 GBF1, DST, PHLDB2

kinetochore 9.75E-4 11.3 5 ANAPC16, CKAP5, INCENP, CLIP1, CENPF chromosome, centromeric region 3.99E-2 9.4 3 INCENP, CENPF, BIRC5

spindle 5.69E-4 8.8 6 SPECC1L, HAUS3, INCENP, CENPF, BIRC5, ATM midbody 7.88E-4 8.2 6 ANKRD54, INCENP, CENPF, BIRC5, MTCL1, TACC1 microtubule cytoskeleton 7.14E-3 6.5 5 HAUS3, CKAP5, CLIP1, DST, TACC1

nuclear envelope 1.24E-2 5.5 5 CHMP7, CLIP1, CENPF, TMPO, DST

cell-cell adherens junction 2.15E-3 4.4 8 LIMA1, CKAP5, DIAPH3, NECTIN2, TMPO, CDH2, SLC9A3R2, PHLDB2

protein complex 2.75E-2 3.0 7 CEP97, EIF4EBP1, CAV1, CKAP5, INCENP, CDH2, SLC9A3R2 centrosome 3.23E-2 2.9 7 CEP97, HAUS3, CKAP5, CLIP1, CENPF, ESPL1, BCL2L1 cytosol 1.57E-5 2.0 38 CEP97, ANAPC16, HAUS3, GDA, CAV1, CPLX1, DPH6, CHMP7,

DIAPH3, TRAPPC2L, NFKB2, BCL2L1, PTTG1, IGF2BP3, STUB1, FTH1, EIF4EBP1, GBF1, CDKN2C, INCENP, IDH2, LSM2, HBD, CKAP5, WNK1, TRIO, CENPF, ESPL1, BIRC5, NPEPPS, ISCU, IFIT1, MTR, TSC2, AMOT, CLIP1, SH3D19, DST

membrane 3.27E-2 1.6 20 CAV1, ZDHHC5, CKAP5, WNK1, PRRC2A, VPS50, HELZ, BCL2L1, CDH2, HLTF, ALDH16A1, TACC1, AQR, GBF1, C1QTNF3, TSC2, TMPO, FAM129A, MDN1, MED1

nucleus 4.31E-3 1.4 44 RPP38, C1ORF174, DPH6, DIAPH3, PRRC2A, HELZ, NFKB2, UBE3C, PTTG1, IGF2BP3, FTH1, GTF2E2, SIN3B, ANKRD54, TMEM57, EDRF1, CDKN2C, EIF1, SUPT5H, MDN1, TRIP11, NFX1, ZNF280C, RAD51AP1, ZNF646, ZNF66, CENPF, ESPL1, BIRC5, ARID1A, NPEPPS, HLTF, SLC9A3R2, TACC1, ATM, ZNF629, ISCU, UACA, TSC2, TMPO, RNF20, DST, SUPT6H, MED1